Dopamine plays an important role as a neurotransmitter in the mammalian nervous system. Indeed, the selective dysregulation of dopaminergic neural transmission has been indicated in a number of neurological disorders including schizophrenia, Parkinson's disease, Huntington's disease, symptoms of attention deficit hyperactivity disorder, Tourette's syndrome, and drug abuse. Virtually all current anti-schizophrenic drugs act as antagonists at a major subclass of dopamine receptors, but are not completely effective and produce undesirable side effects. In addition, cocaine, amphetamines, opiates, nicotine and alcohol have all been shown to be capable of modifying dopaminergic transmission. Therefore, dopaminergic transmission is an important factor in maintaining the mental health of an individual.
A major target for midbrain dopaminergic neurons is the neostriatum. About 95% of all neostriatal neurons have a similar morphology and are referred to as medium-sized spiny neurons. Dopamine- and cyclic AMP (cAMP)-Regulated PhosphoProtein DARPP-32) is a 32 kilodalton cytosolic protein that is selectively enriched in medium-sized spiny neurons in neostriatum [Ouimet et al., Neurosci 4:114-124, (1984); Walaas and Greengard, J Neurosci 4: 84-98 (1984)]. The sequence of Human DARPP-32 has been determined [Brene et al, J. Neuroscience, 14:985-998 (1994); GenBank Accession: AAB30129.1] and a knockout mouse lacking DARPP-32 has been constructed [U.S. Pat. No. 5,777,195, Issued Jul. 7, 1998; Fienberg et al., Science 281:838-842 (1998), the contents of each are hereby incorporated by reference herein, in their entireties].
DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) on a single threonine residue, the thirty-fourth amino acid in the sequence, i.e., Thr34 which results in the conversion of DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP1) [Hemmings et al., Nature 310: 503-505 (1984)]. DARPP-32 can be dephosphorylated at Thr34 in vitro by the calcium/calmodulin-dependent protein phosphatase, calcineurin [King et al., J Biol Chem 259:8080-8083. (1984)]. Dephosphorylation of Thr34 of DARPP-32 removes its inhibitory effect on PP1.
Dopamine has been shown to stimulate the phosphorylation of DARPP-32 in the neostriatum by activation of a biochemical cascade involving stimulation of D1 receptors, activation of adenylyl cyclase, increased cAMP formation and increased activity of PKA [Walaas and Greengard, J Neurosci 4:84-98 (1984)]. The selective enrichment of DARPP-32 in dopaminoceptive neurons and its regulation by dopamine strongly indicate that DARPP-32, through regulating protein phosphatase-1 activity, plays a key role in mediating the effects of dopamine on these cells. Indeed, in the brain the chain of events has been described as the DARPP-32/Protein Phosphatase-1 cascade [Greengard et al., Neuron, 23:435-447 (1999)].
The control of protein phosphatase-1 activity by DARPP-32 is likely to have a significant role in the regulation of neuronal excitability. For instance, in neostriatum, dopamine-mediated effects on the function of calcium channels [Surmeier et al., Neuron 14:385-397 (1995)), voltage-dependent sodium channels [Surmeier et al., Proc. Nat. Acad. Sci., USA 89:10178-10182 (1992); Schiffman et al., Am J Physiol 483:95-107 (1994)] and Na+,K+-ATPase [Aperia et al., Proc Natl Acad Sci, USA 88:2798-2801 (199±1)] are all regulated directly or indirectly by protein phosphatase-1.
Medium-sized spiny neurons of the neostriatum and nucleus accumbens receive dopaminergic input from cell bodies in the midbrain [Anden et al., Life Science 3:523-530 (1964); Poirier and Sourkes, Brain 88:181-192 (1965); Swanson, Brain Res Bull 9: 321-353 (1982)]. To date, five dopamine receptor subtypes have been identified which constitute two major subclasses, a D1 sub family (D1 and D5 subtypes) and a D2 subfamily (D2, D3 and D4 subtypes) [Sibley and Monsma, Trends in Pharmacol Sci 13:61-69. (1992)]. D1 and D2 dopamine receptors are abundantly expressed on cell bodies and dendritic processes of medium spiny neurons [Levey et al., Proc Natl Acad Sci, USA 90:8861-8865 (1993)]. Messenger RNAs coding for each of the other dopamine receptor subtypes (i.e., D3, D4, a nd D5) have been isolated from individual neostriatal neurons [Surmeier et al., J Neurosci 16:6579-91 (1996)], but whether these receptor proteins are expressed in medium spiny neurons and how they functionally interact with D1 and D2 receptors is still unclear.
There is considerable evidence for either synergistic or opposing interactions of D1-like and D2-like dopamine receptors at the biochemical, physiological, and behavioral level [see Jackson and Westlind-Danielsson, Pharmac Ther 64:291-370 (1994) for review]. Biochemically, D1 and D2 receptors have opposing actions on the activity of adenylyl cyclase in neostriatal neurons; whereas activation of D1 receptors increases cAMP formation by adenylyl cyclase, D2 receptors inhibit adenylyl cyclase activity [Stoof and Kebabian, Nature 294: 366-368 (1981)]. Studies have shown that D2-like dopamine receptors via interactions with specific G-proteins, can be coupled to multiple effector systems, including calcium channels, potassium channels and phospholipase C [for review, see Huff, Cell Signal 8: 453-459 (1996)]. For example, Yan et al. [Soc. Neurosci. Abst. 26:1088 (1996)] have shown that D2 receptors on 115 neostriatal neurons negatively couple to calcium channels through a Gi/o class protein. In addition, activation of D2 receptors apparently decreases sodium currents in medium spiny neostriatal neurons through a membrane-delimited pathway and increases these currents through a soluble second messenger pathway (presumably involving inhibition of adenylyl cyclase) [Surrneier et al., Proc. Natl. Acad. Sci., USA 89:10178-10182. (1992)].
Heretofore, there has been no particular link between DARPP-32 and Cdk5, a member of the cyclin-dependent kinases (cdks) [See generally, Sherr, Cell 79:551-555 (1994) and Sherr, Cell 73:1059-1065 (1993)]. However, Cdk5 [also known as neuronal cyclin-dependent-like protein (Nclk) and tau protein kinase II (TPKII)] has been reported to function in cortical lamination, neurite outgrowth, neuronal plasticity, ischemia, apoptosis, myogenesis and in estrogen signal transduction. This kinase has also been shown to be involved in the hyper-phosphorylation of neurofilaments, which form neurofibulary tangles observed in a number of neurodegenerative diseases.
Cdk5 has been designated a member of the cyclin-dependent kinase family based on its high degree of DNA and amino acid sequence homology with other cdks. However, whereas active cyclin dependent kinases consist of a positive regulatory subunit (the cyclin) and a catalytic subunit (the cyclin dependent kinase) Cdk5 atypically can phosphorylate its substrates without the assistance of a cyclin regulatory subunit. Instead, Cdk5 employs a non-cyclin cofactor called neuronal cyclin-dependent-like kinase 5 associated protein (Nck5 a). There are at least two isoforms of Nck5a in the brain (p35 and p39) which may also exist as proteolytic fragments (i.e., p25 and p29, respectively).
Cyclin dependent kinases play an important role during the cellular replication cycle with the regulation of the human cell cycle requiring the periodic formation, activation, and inactivation of protein kinase complexes that consist of a cyclin subunit and a cdk subunit. Indeed, there has been significant interest in cdks in regard to cancer treatment due to the role of cdks in cell division. In contrast, in adults, Cdk5 is not only most highly expressed in the brain, it is also expressed throughout the brain, and furthermore is only active in the brain. Since brain cells are for the most part post-mitotic, i.e., they no longer divide, Cdk5 also is an atypical member of the cyclin-dependent kinase family because it appears to have a role that is independent of cell division.
Heretofore, most of the drugs that are used to treat dopamine-related disorders function at the extracellular surfaces as either D1 receptor agonists or D2 receptor antagonists. These compounds often have a limited period of efficacy and produce unwanted side-effects. Many of these unwanted side-effects are the result of the lack of specificity of the drug for its target. Therefore, there is a ne ed to provide new drugs assays which can be used to develop novel drugs that can be used to treat dopamine-related disorders. Such novel drugs would have great er specificity than those currently used and therefore, would be less likely to have unwanted side-effects. Furthermore, there is a need to develop treatments for diseases/conditions which are due, at least in part, to an aberration or dysregulation of a pathway effected by the neurotransmission of dopamine.
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